VIRAL IMMUNOLOGY

Volume 00, Number 00, 2020

ª Mary Ann Liebert, Inc.
Pp. 1–11
DOI: 10.1089/vim.2020.0082

An Improved DNA Vaccine Against Bovine Herpesvirus-1

Using CD40L and a Chemical Adjuvant Induces Specific
Cytotoxicity in Mice

Cecilia A. Langellotti,1,* Mariela Gammella,1,* Ivana Soria,1 Carolina Bellusci,2 Valeria Quattrocchi,1
Monica Vermeulen,3 Claudia Mongini,1 and Patricia I. Zamorano4
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Abstract

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading
intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are
necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These
vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determi-
nation of the real prevalence of infection. DNA vaccines are a good option because of the capacity of
Differentiating Infected from Vaccinated Animals—(DIVA vaccine)—and may be the best way to induce
cytotoxic responses. Although this type of vaccines leads to only weak ‘‘in vivo’’ expression and poor immune
responses, incorporation of molecular and/or chemical adjuvants can improve the latter, both in magnitude
and in direction. In this study, we have investigated the specific immune responses elicited in mice by DNA
vaccines based on the BoHV-1 glycoprotein D (pCIgD) with and without two different adjuvants: a plasmid
encoding for murine CD40L (pCD40L) or Montanide 1113101PR (101). Mice vaccinated with
pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 developed significantly higher specific antibody titers
against BoHV-1 than the pCIgD group ( p < 0.01). The animals vaccinated with pCgD+pCD40L+101 raised
significantly higher levels of IgG2a and IgG2b ( p < 0.01 and p < 0.001, respectively) than mice vaccinated with
pCIgD alone. On the contrary, when the activity of CTL against cells infected with BoHV-1 was measured, the
vaccine pCgD+pCD40L+101 induced significantly higher levels of cytotoxicity activity ( p < 0.001) than pCIgD
alone. A significant increase in the CD4+ populations in the group receiving pCIgD+CD40L+101 in comparison
with the pCIgD group was observed and, also, interferon gamma, interleukin (IL)-6, and IL-17A levels were
higher. Considering the results obtained from this study for humoral and cellular responses in mice, the
inclusion of pCD40L and 101 as adjuvants in a BoHV-1 DNA vaccine for cattle is highly recommendable.

Keywords: BoHV-1 DNA vaccine, CD40L, chemical adjuvants, cytotoxic response, mice model

Introduction abortion. More importantly, infected animals that are im-

munosuppressed may suffer secondary bacterial infections

B ovine herpesvirus-1 (BoHV-1) is an important viral

pathogen of cattle, and it produces a variety of diseases,
including vulvovaginitis/balanoposthitis, infectious bovine
rhinotracheitis, systemic infection in the newborn, and
abortion in cows (35,73). The economic impact of this in-
fection related to weight loss, decreased milk production, and
increasing morbidity and mortality (21,36).
However, if infection or reactivation of the virus occur
during pregnancy, abortions can occur (54,59,70). Six Eu-
ropean countries became BoHV-1 free after the introduction
of expensive eradication programs, consisting of seroposi-
tive animals culling (62,71).

1
Instituto de Virologı́a-IVIT (INTA-CONICET), Buenos Aires, Argentina.
2
Universidad Nacional de Rio Negro, Sede Atlántica, Viedma, Rı́o Negro, Argentina.
3
Laboratorio de células presentadoras de antı́geno y respuesta inflamatoria. Instituto de Medicina Experimental (IMEX) - CONICET,
Academia Nacional de Medicina, Buenos Aires, Argentina.
4
Cátedra de Inmunologı́a Aplicada, Universidad del Salvador, Buenos Aires, Argentina.
*These authors contributed equally to the results presented in this report.

1
 2 LANGELLOTTI ET AL.
A more common way to prevent BoHV-1-related dis-
eases is the use of inactivated or attenuated live vaccines.
However, they cannot control latent infection. The ability to
differentiate infected from vaccinated animals (DIVA) is an
important issue (65). Live-attenuated vaccines may cause
abortion, latency with reactivation and shedding (79,80).
Also, they may downregulate major histocompatibility
complex (MHC) class I molecules (31,55), which com-
promise the subsequent cellular immune response. On the
contrary, inactivated viral vaccines have a short duration of
immunity and are poor inducers of cellular responses (17).
Importantly, BoHV-1 can persist in the presence of antiviral-
specific antibodies (24,38,52). So, cytotoxic T lymphocytes
(CTLs) are necessary for controlling the virus (73). Novel
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vaccines against BoHV-1 that do not have the problems as-
sociated with current vaccines are thus necessary. They need
to contain viral epitopes capable of inducing protective im-
mune responses, and should ideally be suitable for DIVA.
DNA vaccines have potential to induce cell-mediated and
humoral immune responses with the advantages of having
good thermostability, the capability of coexpression of
multiple antigens and reduced production costs since they
avoid the need of working with viral particles (4,10,28).
Licensed DNA vaccines exist for hematopoietic necrosis
virus in salmon (Apex-IHN, Novartis) (26), West Nile virus
in horses (West Nile Innovator, Fort Dodge) (15), and
melanoma in dogs (Canine Melanoma Vaccine, Merial) (6).
Although genic vaccines have the capacity to induce
cytotoxic responses (40), they have limited potency because
they do not amplify or propagate ‘‘in vivo.’’ For this reason,
it is necessary to increase the magnitude and direction of the
immune response with the incorporation of molecular and
chemical adjuvants.
Viral glycoproteins are involved in several steps of
BoHV-1 replication (7,25,43,57). Glycoprotein D (gD) is
responsible for the penetration of the BoHV-1 in cells with
participation in the membrane fusion and viral adsorption
(74). It induces neutralizing antibodies (3,13,17) and has
cytotoxic epitopes (16,17). Notably, vaccination with a
plasmid encoding the secreted form of the gD encoding in a
vector produced a different immune response (type 1/type 2)
than vaccination with one encoding full-length gD (41,42).
Injection of a naked plasmid vector into animal muscle or
skin can induce an immune response against a transgene-
encoded antigen (Ag) (60), but in general this response is
weak and needs to be enhanced to provide protective im-
munity (23,49,61). The interaction between the CD40 re-
ceptor and its ligand, CD40L, regulates humoral and cellular
immune responses. CD40L (or CD154) is a 39-kDa type II
membrane glycoprotein with homology to TNF-a (2). This
molecule is expressed transiently on CD4+ T cells within
hours after ligation of the TCCRD 3 complex and then is
rapidly downmodulated (66). CD40L allows the T lympho-
cyte to interact with B lymphocytes, monocytes, and den-
dritic cells (DCs), inducing their proliferation, differentiation,
and expressing accessory molecules that facilitate the pre-
sentation of antigens to T lymphocytes (22). Given the ability
of CD40L to enhance the development and proficiency of
antigen-presenting cells (APCs), we hypothesized that coin-
jection of a DNA vaccine with a plasmid encoding CD40L
could improve the immune response to the transgene antigen.
Inoculation of a plasmid coding for bovine CD40L fused to a
secreted form of BoHV-1 gD, in sheep, enhanced the humoral
immune response to a plasmid coding for gD alone (47). On
the contrary, immunized calves with the same vaccine had
significantly higher gD-specific serum titers of immuno-
globulins G and A than plasmid gD vaccine alone, although
protection upon BoHV-1 challenge was not achieved (48).
Mice have been used as an experimental model to select
optimal formulations for BoHV-1 vaccines before carrying
out experiments in large animal hosts (67).
We have previously shown in the mouse model that in-
corporation of adjuvants into a genetic vaccine against
BoHV-1 is immunologically beneficial (18,40). Recently, we
demonstrated that bovines vaccinated with a DNA vaccine
coding BoHV-1 gD, adjuvanted with Montanide 1113101PR
(101), induced better protection upon challenge than animals
vaccinated with a genic vaccine alone. Although this vaccine
was able to diminish clinical symptoms and viral excretion,
our results showed that it is still necessary to induce a higher
immune response against the virus (61).The 101 adjuvant is
composed of non-crosslinked charged polymers (10–500 nm)
dispersed in water with an immunostimulating compound. It
has the capacity to improve DNA binding and transfection
potency. Also, it can recruit cells to the site of injection (78).
In this study, we evaluate the immune response induced
in mice by a DNA vaccine containing the truncated, secreted
version of BoHV-1 gD (18), in combination with the adju-
vants CD40L and 101, to extend our previous studies in the
murine model.
Materials and Methods
Plasmid construction
Polymerase chain reaction (PCR) product encoding the
secreted version of the BoHV-1 glycoprotein D (gD) was
inserted into EcoRI-digested pCIneo vector (Promega) to
construct plasmid pCIgD, prepared as described before (40).
Plasmid pDNA3.1 contained the gene for murine CD40L
(pCD40L) and was kindly provided by Dr. Kipps, University
of California. Nonrecombinant plasmid pCIneo was used as
control. All plasmid preparations had A260/280 ratios >1.7.
After purification, plasmids were diluted with phosphate-
buffered saline (PBS).
Adjuvant
Montanide 1113101PR (101) was provided by Seppic
Inc., France, and used according to the manufacturer’s
instructions.
Toxicological tests made on Montanide adjuvants (derml
irritation test, Oral LD 50, Berlin test, IP LD 50, pyroge-
nicity, ocular irritation test) can be considered nontoxic and
well tolerated. The Committee for Veterinary Medical
Products (CVMP) considered these adjuvants safe for use in
immunological products. These substances have been au-
thorized in the annex of the European Council Regulation n
470/2009 (previously 2377/90/EC).
Generation of bone marrow-derived DCs
Bone marrow-derived DCs were obtained as previously
described (39). In brief, the marrows of femurs and tibiae of
naı̈ve BALB/c mice (n = 3) were flushed out with RPMI
1640 medium. After washing, cells were diluted at 1 · 106
 CYTOTOXIC ACTIVITY USING A DNA VACCINE
cells/mL in RPMI 1640 medium (Invitrogen Life Technol-
ogies) with 10% fetal calf serum (FCS), 5.5 · 10 - 5 of 2-
mercaptoethanol (Sigma-Aldrich), and 30% conditioned
medium from GM-CSF-producing NIH-3T3 cells. The cells
were cultured for 9 days with 50% of the medium replaced
every 2 days. On day 9 of culture, >85% of the harvested
cells expressed CD40, CD11c, MHC class II, and CD80 but
no Gr-1 (data not shown).
Endocytosis assay
DCs (produced from naı̈ve mice) were incubated with
medium, pCIneo (1 lg/mL), pCIgD (1 lg/mL), 101 adjuvant
(ratio 24% adjuvant and 76% PBS), and pCIgD+101 (ratio
24% adjuvant and 76% DNA). Then, 100 lg/mL FITC-
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labeled OVA was added to RPMI 1640 medium and incu-
bated for 40 min at 37C. After incubation, the cells were
washed with cold PBS three times, and evaluation of uptake
was tested by flow cytometry. The fluorescence background
in cells incubated with FITC-OVA at 4C was determined
and subtracted. The results are expressed as mean fluores-
cence intensity values.
Animal treatment and vaccine formulations
Male BALB/c mice (28 – 2 g) from La Plata University
(Argentina) were used. The animals were acclimated and
randomly distributed into five experimental groups. Mice
were kept in rodent caging with ventilated systems, and re-
ceived water and food ad libitum. Handling and housing of
animals were according to the guidances of the Institutional
Committee for Care and Use of Experimental Animals,
CICUAE-INTA, Argentina (Approved Protocol n 54/2016).
Groups of 5 mice were intradermically (i.d.) inoculated in
the back, at days 0 and 15, with 15 lg of pCIgD and 75 lg
of pDNA3.1-mCD40L (pCD40L) formulated without and
with Montanide 101 adjuvant in a final volume of 0.2 mL
(distributed in two sites) (The dose of plasmid injected
was previously selected based on dose-response curve). The
same amount of pCIneo was inoculated as negative control
group. Serum samples were obtained at days 14 and 30.
Virus
BoHV-1 strain LA (Los Angeles) was propagated in
Madin Darby bovine kidney (MDBK) cells, in E-MEM
medium supplemented with 10% fetal bovine serum (Gib-
co). The virus was added at a multiplicity of infection (moi)
of 0.1, and it was allowed to adsorb for 45 min at 37C
before the addition of more culture medium. The superna-
tant was collected, and cell debris was removed by centri-
fugation at 23,699 g for 1 h at 4C, when cytopathogenic
effects were observed. Supernatants of infected cells were
pelleted by ultracentrifugation at 120,000 g for 1 h at 4C.
The virus suspension was stored at -70C. Inactivated virus
(30 min at 11 cm from two General Electric G875 ultraviolet
bulbs) was used for enzyme-linked immunosorbent assay
(ELISA) and in vitro cell stimulation.
Assembly of gD expression vector and production
of recombinant gD
The sequence coding for full-length gD was amplified by
PCR from BoHV-1 DNA, using the following forward and
3
reverse primers: 5¢-AAGAATTCATGTTGCCTACACCCG
CGCCGCGGGT-3¢ and 5¢-AAGAATTCTCAGGCGTCGG
GGGCCGCGGGCGTA-3¢ that contain EcoRI restriction
sites and amplify a 1005 bp fragment (nt 138 to 1143) of the
gD gene (emb AJ004801 BHV1CGEN). The ATG start codon
for expression in bacteria is provided by the pRSETB vector
before a 6 His-codon stretch, while the reverse primer includes
the gene stop codon. The 1005 bp fragment was inserted into
plasmid pRSET-B (Invitrogen) to produce pRSETgD. The
correct orientation and the integrity of the gD sequence were
confirmed by nucleotide sequencing and restriction analysis
(data not shown). E. coli BL21 cells were used to express the
recombinant plasmid by standard methods. Expression was
induced with 1 mM IPTG (Sigma) for 2 h when the absor-
bance was 0.3 at 600 nm. Proteins were extracted with 6 M
guanidine HCl and 0.5 M NaCl (denaturing buffer), and gD-
his protein was purified by Ni-nitrilotriacetic acid (Ni-NTA)
Agarose columns (Invitrogen).
ELISA for detection of anti-BoHV-1 antibodies
Immulon 1 plates were coated with 50 lL of inactivated
BoHV-1 (iBoHV-1) (2 · 107 TCID50/mL) in 0.1 M
carbonate-bicarbonate buffer, pH 9.6. The plates were incu-
bated overnight (ON) at 4C. They were blocked, and serial
dilutions (1:10) of mice sera were diluted in PBS/0.05%
Tween 20 (PBST) containing 1% ovalbumin (PBST-OVA)
and dispensed in 50 lL/well aliquots. After incubation, plates
were washed with PBST, and antimouse IgG peroxidase
conjugate (KPL) was added for 1 h at 37C. Finally, ortho-
phenylene-diamine (1,2-benzenediamine) dihydrochloride
(OPD; SIGMA) and H2O2 were added, and absorbance was
measured at 492 nm in a MR 5000 microplate reader (Lab-
systems, MN). The cutoff was established as the mean A492
of the negative sera +2 standard deviations. Titers were ex-
pressed as the log10 of the reciprocal of the highest serum
dilution giving an A492 higher than the cutoff.
IgG1, IgG2a, IgG2b, and IgG3 isotyping ELISA
Greiner microtiter plates were coated with purified re-
combinant gD (1,4 lg/well) to evaluate the IgG1, IgG2a,
IgG2b, and IgG3 subtypes in vaccinated mice. Plates were
blocked with PBST containing 0.5% gelatine (PBST-G), and
then serial dilutions (1:10) of mice sera were prepared in
PBST-G and added to plates in 50 lL/well aliquots, for 2 h
at room temperature. PBST was added to wash the plates.
Biotinylated goat antimouse IgG1, IgG2a, IgG2b, and IgG3
(Caltag Laboratories, San Francisco, CA) were dispensed
and after 60 min incubation, plates were washed, and anti-
mouse IgG peroxidase conjugate (KPL) was added and in-
cubated for 1 h at room temperature. The reaction was
visualized as described in section ‘‘ELISA for detection of
anti-BoHV-1 antibodies.’’
Percentage of BoHV-1 neutralization
When seroneutralizing antibodies were measured in sera
of immunized mice at 30 dpv using 100 DICT50 infective
tissue culture doses, all values were negative. So, it was
decided to measure the percentage of neutralized virus in-
cubated 1/8 serum dilution at 25 lL of volume with different
fivefold dilutions of infective BoHV-1 (1000 to 1 TCID50
 4 LANGELLOTTI ET AL.

per 25 lL) in quadruplicates. Then, the virus–serum mix-
ture, previously incubated ON at 37C under 5% CO2, was
seeded on MDBK cell monolayers. After 1 h at 37C, cells
were washed with PBS. Finally, cells were incubated with
fresh medium supplemented with 2% FCS at 37C under 5%
CO2. After 72 h, cytopathic effects were observed. The
remnant infective virus was determined by a TCID50 assay.
The calculated viral titer was expressed as the percentage of
BoHV-1 that was still infective after serum incubation,
compared with the titer of virus suspensions incubated
without serum (a 25 lL volume of medium was incubated
with the different dilutions of infective BoHV-1).
Cytokines detection
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In vitro CFSE proliferation assay
Carboxylfluorescein diacetate succinimidyl ester (CFSE;
5 nM) (BD Biosciences) was used to label splenocytes
for 40 min at 37C. Cells were washed four times and re-
suspended in RPMI complete medium. The cells were cul-
tured with medium, iBoHV-1 or Concanavalin A for 4 days.
Then cold PBS was used to wash the splenocytes. Fixation
was done by resuspension in 0.2% paraformaldehyde in
FACSFlow (BD, Buenos Aires, Argentina) and analyzed
by cytometry.
Statistical analysis
Data between three or more groups were analyzed using

one-way analysis of variance (ANOVA) and Dunnett’s post-

Spleens were extracted from mice, and splenocytes were
tests. Differences were considered significant at p-value <0.05.
disaggregated by gentle homogenization in RPMI 1640
medium/10% FCS (Gibco)/1% ampicillin-streptomycin, 15
days after the booster (30 dpv). In 96-well flat-bottomed Results
plates, pool of splenocytes from each group were plated pCIgD plus 101 adjuvant enhances
(1 · 106 cells per well) in 100 lL complete medium [RPMI endocytosis effect on DCs
1640/1% ampicillin-streptomycin/1 mM sodium pyruvate
(Gibco)/10% FCS/5 mM HEPES (Gibco)/2 mM L-glutamine Immature DCs are specialized to sense the microenviron-
(SIGMA)/50 mM 2-mercaptoethanol/10 mM MEM Non- ment and incorporate the antigen through phagocytosis or
essential Amino Acids Solution (Gibco)]. In triplicate 100 lL endocytosis. With the aim to determine if the selected adju-
of inactivated BoHV-1 was added to the wells in complete vant for the genic vaccine or the DNA vaccine were able to
medium or medium alone, and incubated for 48 h at 37C. affect the antigen uptake by endocytosis in naı̈ve DCs, these
The supernatant was collected, and interferon gamma (IFNc), cells were cultured in vitro with pCIneo, pCIgD, pCIgD+101,
interleukin (IL)-4, IL-6, IL-17A, and IL-10 were measured 101 adjuvant alone, or medium like a basal control. Finally,
using BD CBA Kits (Mouse Th1/Th2/Th17) by flow cy- cells were incubated with OVA coupled to FITC for 40 min at
tometry. The results were expressed in pg/mL. 37C. Since this is a temperature-dependent phenomenon, the
reaction control was DCs incubated with OVA-FITC at 4C.
Endocytosis levels were then measured by flow cytometry.
Cytotoxic T cell assay ( JAM test)
The 101 adjuvant plus pCIgD produced a significant in-
Cytotoxicity assay was previously described by Matzin- crease of endocytosis in these cells. On the contrary, pCIgD,
ger et al. (50). In brief, splenocytes were isolated as de- pCIneo, or 101 adjuvant alone did not modify endocytosis
scribed in section ‘‘Cytokines detection.’’ Cells were levels (Fig. 1).
cultured with inactivated BoHV-1 or medium alone for 5
days at 37C and 5% CO2. P815 cells infected with a moi of
5 of BoHV-1 and labeled with [methyl-3H] thymidine were
used as target cells. 104 P815 cells per well were plated with
dilutions of effector cells (splenocytes) and incubated in
triplicate for 3 h at 37C. The effector/target ratio used in the
assay was 80:1. A semiautomatic cell harvester (Skatron)
was used to harvest the cells. Radioactivity was measured
by liquid scintillation counting (experimental release).
Spontaneous release was determined in target cells incu-
bated with complete medium only. Percentages of specific
lysis were calculated using the formula:
% Specific lysis = 100 · [(spontaneous release – experi-
mental release)/spontaneous release].

Immunofluorescent staining
The following labeled monoclonal antibodies were used
to stain the surface markers of splenocytes: allophycocyanin
anti-CD8b, fluorescein isothiocyanate antimouse CD4, and
conjugated rat Ig isotype controls (Pharmingen). Spleno-
FIG. 1. Adjuvant effect on BMDC. Endocytosis of OVA-
cytes were incubated with labeled antibodies for 20 min at FITC antigen by BMDC cultured in vitro with different
4C. Then, they were washed with PBS containing 1% FCS formulations for 48 h was tested. Results are expressed as
and fixed with 0.2% paraformaldehyde. The stained cells the MFI – SEM of five independent experiments. BMDC,
were evaluated by flow cytometry in a BD FacsCalibur and bone marrow-derived dendritic cells; MFI, mean fluores-
analyzed with CellQuest software. cence intensity; SEM, standard error of the mean. *p < 0.05.
 CYTOTOXIC ACTIVITY USING A DNA VACCINE
In previous studies, cattle were immunized with pCIgD
and adjuvant 101. After viral challenge, higher protection
levels were observed as compared with animals immunized
with pCIgD alone (61). For this reason, adjuvant 101 was
selected for this study, with the addition of a plasmid en-
coding CD40L, to enhance the immune response against gD.
pCIgD vaccines formulated with pCD40L, 101
adjuvant, and pCD40L plus 101 elicit higher specific
humoral immune responses than a pCIgD vaccine.
Neutralizing capacity of the sera
Groups of mice were vaccinated with 15 lg of pCIgD (per
dose) formulated with and without pCD40L, 101 adjuvant,
and a combination of both, and the elicited immune re-
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sponses were evaluated and compared. Analysis of sera by
ELISA to BoHV-1 showed that at 14 dpv, all animals in
groups immunized with pCIgD+pCD40L, pCIgD+101, and
pCIgD+pCD40L+101, were able to induce specific BoHV-1
antibodies. Moreover, at 30 dpv, levels of antibodies against
BoHV-1 in groups pCIgD+pCD40L, pCIgD+101, and
pCIgD+pCD40L+101 were significantly higher than those in
the pCIgD group ( p < 0.01). None of the pCIneo-immunized
control animals produced any specific immune response at
any time compared with normal mouse serum (Fig. 2A).
Moreover, analysis of sera by ELISA showed that specific
antibodies against the gD-protein were induced at 14 dpv.
Total antibody levels were significantly increased ( p < 0.05)
at 14 dpv in the pCIgD+101 group compared with the pCIgD
group. In contrast, at 30 dpv all immunized groups showed
the same high antibody titers (Fig. 2B).
Increases in IgG isotype induced by the different vaccines
were studied next. At 14 dpv, IgG2a and IgG2b were the main
isotypes in the sera of mice vaccinated with pCIgD+CD40L,
and were significantly increased with respect to the pCIgD
group (IgG2a p < 0.05; IgG2b p < 0.01). The same was ob-
served in the group immunized with pCIgD+101 adjuvant
(vs. pCIgD: IgG2a, p < 0.05; IgG2b, p < 0.001) and with
pCIgD+pCD40L+101 adjuvant (vs. pCIgD: IgG2a, p < 0.01;
IgG2b, p < 0.001). Furthermore, group pCIgD+101 presented
significantly higher levels of specific IgG1 ( p < 0.05) than the
pCIgD group. At 30 dpv, although specific gD isotype titers
were high, IgG1 and IgG3 showed higher levels, and no sig-
nificant differences between groups were observed (Fig. 2C).
The viral neutralizing capacity of sera from immunized
animals was studied in a 1/8 dilution. At 30 dpv (Fig. 2D), the
average percentages of neutralized virus were 1.8 for sera in
the pCIneo group, 18.4, 35.7, 55.7, and 24.4 for the pCIgD,
pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 group,
respectively. However, when antibodies were evaluated in a
neutralization test with a fixed amount of virus, no serum
sample at 30 dpv was able to neutralize 100 TCID50 of
BoHV-1 (virus neutralization titers <0.9, data not shown). This
assay evidenced a weak capacity of the antibodies to neutralize
virus in all animals. However, a differential neutralizing capacity
was observed among groups, being the animals immunized with
pCIgD+101 those that managed to neutralize the largest amount
of viral particles in comparison to the other groups.
Cytokine production
To profile the cytokine levels induced by different vac-
cines at 30 dpv, production of some cytokines was evaluated
5
by cytometric bead arrays on a pool of supernatant of
splenocytes stimulated or not with inactivated BoHV-1 or
concanavalin A, as a positive control, from each group of
vaccinated mice.
The pCIgD vaccine group secreted lower levels of IFNc,
IL-4, IL-6, IL-17, and IL-10 cytokines than the other for-
mulations containing pCIgD (Fig. 3A–E).
The amount of IFN-c produced upon vaccination with
pCIgD+pCD40L (694.64 pg/mL) was *20 times higher
than the amount produced upon vaccination with pCIgD
alone (32.49 pg/mL), and 11 times higher than those in the
pCIgD+101 or pCIgD+pCD40L+101 groups (Fig. 3A).
Also, in the case of IL-6 and IL-10, pCIgD+pCD40L group
is the major producer of both cytokines as compared with
the other groups (Fig. 3B, C). To highlight in this sense
that the pCIgD+pCD40L+101 group secrete similar levels
of IL-6 than the pCIgD+pCD40L group. In case of IL10,
the pCIgD+pCD40L group was the only group that pro-
duces this cytokine (Fig. 3C).
Besides, low levels of IL-4 were detected among the
pCIgD, pCIgD+pCD40L, and pCIgD+pCD40L+101 groups,
pCIgD+101 being the major producer group of this cytokine
(Fig. 3D).
Interestingly, production of IL-17A could be detected in
the pCIgD+pCD40L, pCIgD+101, and pCIgD+pCD40L+101
groups but not in the pCIgD one (Fig. 3E). Animals im-
munized with pCIneo only secreted low levels of IL-6.
Combination of pCIgD with pCD40L and 101 adjuvant
increase splenic CD4 + T lymphocytes cell population
Spleen cells obtained from vaccinated mice were used to
determine the effect of these vaccines on T lymphocyte
populations.
A significant ( p < 0.05) expansion of CD4+ T cells popu-
lation was observed at 30 dpv in the pCIgD+101+pCD40L
group as compared with pCIneo and pCIgD (Fig. 4A). No
significant differences, on the contrary, were observed in the
CD8+ lymphocyte populations measured (Fig. 4B). In the other
groups, no changes were observed in these two populations.
Vaccination with pCD40L +101 enhances the specific
cytotoxic T cell in vivo response against BoHV-1
Using a specific viral cytotoxicity test ( JAM test), sple-
nocytes of pCIgD mice with or without pCD40L and/or 101
adjuvant and pCIneo groups were restimulated in vitro with
UV-inactivated BoHV-1 and then incubated with radioac-
tively labeled BoHV-1-infected P815 cells.
CTL activities in the three groups that received pCIgD
plus pCD40L and/or 101 adjuvant, which were restimulated
in vitro with iBoHV-1, were significantly higher
(pCIgD +pCD40L, 21.12% p < 0.01; pCIgD+101, 18.04%
p < 0.05; pCIgD+pCD40L+101, 26.22% p < 0.001) than those
in the pCIgD group (6,95%) (Fig. 5). However, when a cutoff
was selected (pCIneo mean – 2DS = 20% specific cytotoxicity)
only pCIgD+pCD40L+101 had all animals over the cutoff with
specific positive cytotoxicity activity. In contrast, only one and
two animals were positive in the pCIgD+pCD40L and the
pCIgD+101 groups, respectively. These results indicate that the
gD-encoding plasmid plus pCD40L, 101 adjuvant, or both
induce specific CTL that recognizes BoHV-1-infected cells.
Yet, only combined with 101 and pCD40L, pCIgD has the
 6 LANGELLOTTI ET AL.
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FIG. 2. Humoral immune response elicited. (A) Humoral response in mice vaccinated with different formulations. Mice
were intradermally immunized on days 0 and 15, and serum IgG antibody levels specific for BoHV-1 were measured at 14
and 30 dpv. Bars represent the mean of log10 antibody titers from mice in each group – SEM. Significant differences with
respect to the pCIgD group are indicated as **( p < 0.01). (B) gD-specific antibodies detected by ELISA at 14 and 30 dpv.
The results are presented as the mean – SEM of log10 antibody titers. Significance of differences with respect to the pCIgD
group are indicated as *p < 0.05. (C) Anti-gD-specific immunoglobulin isotypes measured by indirect ELISA at 14 and 30
dpv. Data represent the mean – SEM of log10 antibody titers. Significant differences from the mean of the pCIgD group are
indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Neutralization of BoHV-1 by sera from immunized animals. A 1:8
serum dilution from each group (pCIneo, pCIgD, pCIgD+101, pCIgD+CD40L, and pCIgD+CD40L+101) was incubated with
different BoHV-1 doses, and the remnant infective virus was determined by a TCID50 assay. Titer reductions are expressed
as % neutralized BoHV-1 (black bars), and the remnant infective virus is expressed as % non-neutralized BoHV-1 (white
bars). BoHV, bovine herpesvirus-1; ELISA, enzyme-linked immunosorbent assay.

capability to achieve consistently high specific cytotoxicity Discussion

toward BoHV-1 in all animals of the group.
Vaccination with pCIgD +pCD40L or 101 adjuvant
enhances BoHV-1 specific T cell proliferation
Since the cellular immune response is essential to control a
BoHV-1 infection, specific T cell proliferative responses
were measured. As shown in Table 1, there are more animals
with a viral-specific cell proliferative response considered as
positive (above the cutoff = 3.64) in the pCIgD+101 and
pCIgD+pCD40L groups (60% positive proliferation in both
groups against 25% in the pCIgD group). Surprisingly, under
these experimental conditions, no specific proliferation was
detected in the pCIgD+pCD40L+101 group. All splenocytes
stimulated with concanavalin A had a proliferative response.
The major problem to control BoHV-1 resides in the ca-
pability of the virus to produce latency and immune evasion
in infected animals. During infection, the virus propagates
intracellularly followed by an extracellular dispersion, and
persists even in the presence of neutralizing antibodies. Thus,
BoHV-1 vaccines should induce humoral and cellular im-
mune responses with induction of CTL (44,46).
Huang et al. (32) used a plasmid coding BoHV-1 gly-
coprotein B (gB) to induce cellular and humoral immune
responses in mice, and showed production of specific anti-
bodies and CTL responses. The same vaccine induced hu-
moral responses and elicited IFNc secreting CTL in calves
(32). This was the first demonstration that the plasmid in-
duced CTL not only in mice but also in calves. The most
important glycoprotein in BoHV-1 is glycoprotein D (gD),
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FIG. 3. Cytokine detection with CBA. Splenocytes were stimulated or not for 48 h with inactivated BoHV-1 or conca-
navalin A, as a positive control, and cytokine production was detected using a Cytometric Bead Array (CBA) kit. (A) IFNc,
(B) IL-6, (C) IL-10, (D) IL-4, (E) IL-7A. Each bar represents a pool of supernatants of splenocytes from each mouse group.
IFNc, interferon gamma; IL, interleukin.

FIG. 4. Cellular spleen subpopulations from vaccinated mice at 30 dpv. Each point corresponds to one animal of the
group. Results are expressed as population percentages (means – SEM) (A) CD4 lymphocytes (CD4+); (B) CD8 lympho-
cytes (CD8b+). *p < 0.05.

7
 8 LANGELLOTTI ET AL.
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FIG. 5. Immunization with pCIgD and adjuvants induces an enhanced CTL response as compared with pCIgD alone.
Splenocytes collected at 30 dpv and restimulated in vitro with BoHV-1, and were tested for CTL response by a cytotoxicity
assay against the virus. Representative results of percentage specific lysis are shown at an effector/target ratio of 80:1.
*p < 0.05, **p < 0.01, and *** p < 0.001 (statistical significance of differences with respect to the pCIgD group). A cutoff of
20 was calculated as the mean of the pCIneo group +2 SD. CTL, cytotoxic T lymphocytes.

which is involved in the protective immune response (3,33),

Table 1. T Cell Proliferation in Response
to Vaccination with pClgD Alone
and Formulated with 101 and/or CD40L
Treatment N animal % Proliferation*
pClneo 1 0.56
2 1.78
3 2.94
4 1.38
pClgD 1 3.02
2 0.16
3 3.52
4 4.35a
pClgD+pCD40L 1 6.59
2 5.80
3 2.83
4 2.01
5 3.75
pClgD+101 1 6.51
2 3.79
3 1.31
4 3.96
5 0.60
pClgD+pCD40L+101 1 0.10
2 0.97
3 2.52
4 1.64
5 0.51
Spleen cells were isolated from animals inoculated with pCIgD
with adjuvants and in vitro stimulated with iBoHV-1 for 4 days,
followed by measurement of T cell proliferation by lost of CFSE.
* % Proliferation = % proliferation stimulated with BoHV-1 - %
proliferation stimulated with medium.
a
Results in bold and underline are over the cutoff. The cutoff is
calculated as pClneo mean + 2 SD (cutoff = 3.64).
BoHV, bovine herpesvirus-1; SD, standard deviation.
with major targeting to CD8+ CTL (16,17,40), and with the
production of neutralizing antibodies (3,17,33,58,76,77,82).
In this study, a secreted form of BoHV-1 gD was chosen
because previous studies of others performed in mice and
cattle showed that this truncated version of gD induced
higher antibody titers than the membrane-anchored form
(41,42,75).
Given the importance of adjuvants to increase vaccine
efficiency, the immunomodulatory effects on a DNA vac-
cine of Montanide 1113101PR (101) adjuvant (SEPPIC)
were previously analyzed in our laboratory. We demon-
strated that a DNA vaccine based on BoHV-1 gD formu-
lated with 101 adjuvant induced not only specific antibodies
but also cellular immunity against BoHV-1 with viral-
specific T cell proliferation and the high levels of IFNc
secretion in cattle (33).
This study examined the benefits of the CD40 ligand,
CD40L. This molecule belongs to the tumor necrosis
factor superfamily, hasa transient expression in activated
T lymphocytes, and can stimulate DCs and B cells (27).
This stimulation activates DCs and primes the CD8+
T cell response (5,64,68). Also, CD40L increases the
survival and differentiation of activated B cells (20).
These qualities make CD40L an attractive vaccine adju-
vant (8,30,34,51,69,81).
Incorporation of any of these adjuvants (101, pCD40L,
or both) to gD vaccine formulations yielded an increased
humoral response toward BoHV-1 and gD as well. Similar
results for 101 and CD40L were reported by Quattrocchi
et al. (61) and Manoj et al. (48), respectively. Vaccines
formulated with these adjuvants yielded high levels of
antibodies in cattle, but insufficient protection upon
BoHV-1 challenge.
Analysis of isotypes in serum showed that IgG1, IgG2a,
IgG2b, and IgG3 levels were similar in all animals, but the
major variation was observed in IgG2a and IgG2b at 14 dpv.
 CYTOTOXIC ACTIVITY USING A DNA VACCINE
An increase in IgG2a has been described for DNA vaccines
(63) and suggests a T-helper 1 response. Predominance of
IgG2a has been described for humoral protective immune
responses to virus in mice. In these animals, this isotype is
clearly more efficient at complement fixation (11,12,37,56)
and as opsonizing agent than IgG1 (1). Detection of lym-
phocyte proliferation after inactivated BoHV-1 stimulation
and isotype switch evidenced by IgG2a secretion indicates a
balance to a Th1/Th2 profile of CD4+ T cells (11,12,53,72).
One feature of adjuvants is their inflammatory effect that
attracts inflammatory cells, including macrophages and DCs
(14,78). This characteristic plays an important role in the
immune response. On the contrary, the formulation of
pCIgD with 101 adjuvant can be efficiently endocytozed by
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APCs, generating an increase in the proliferative response of
other cells (29). It can be hypothesized that in the present
experiments the 101 adjuvant recruited APCs at the site of
inoculation, facilitating DNA internalization, as observed by
Dupuis and collaborators (19).
Since cellular immune response is important to deal with
BoHV-1 infection, the specific T cell proliferative response
was measured. Our results showed that this response was
incremented in the pCIgD+101 and pCIgD+pCD40L groups
(60% positive proliferation in both groups against 25% in
the pCIgD group). Surprisingly, under these experimental
conditions no specific proliferation was detected in
pCIgD+pCD40L+101 group. This could be due to expen-
sive cell death after 4 days of proliferation, when cells
were harvested.
Genetic immunizations were used in several animal
models and hosts, such as mice and cattle (9,13,61,75). It
has been demonstrated by Deshpande et al. (17) that in-
duction of specific CTL against BoHV-1 was elicited by
membrane-anchored gD. The same phenomenon was
demonstrated by our group inoculating a DNA vaccine
encoding a secreted version of gD (40). The pCIgD vac-
cine was formulated with 101 and pCD40L with the ex-
pectation of inducing CTL against the virus, and a specific
cytotoxicity assay was set up with BoHV-1-infected P815
cells. This experiment demonstrated that the mice vacci-
nated with different formulations had the capacity to elicit
CTL production. We observed the occurrence of memory
CD8+ cells that recognized and lysed P815-infected cells.
The specific cytotoxicity was consistent with the increases
in IL-17A detected in pCIgD+101, pCIgD+pCD40L, and
pCIgD+101+pCD40L. IL-17A is required for optimal
specific CD8+ T cells activity. In addition to Th17 cells,
there are other sources of IL-17, such as Tcd, NKT, and
CD8+ T cells (45,83). In DCs, IL-17A upregulates surface
MHC class I molecules and enhances production of IL-6.
This facilitates crosspriming and proliferation of CTL. In
the same sense, secretion of IFNc indicates the activation
of a Th1-type response.
Taking together, our results indicate that formulation of a
pCIgD DNA vaccine with adjuvants 101 and/or pCD40L
elicits a higher humoral and cellular immune response
against this virus than the plasmid alone in mice. They also
show that only when both adjuvants are included, CTL is
increased in all immunized animals. Because of this, we
suggest that a pCIgD vaccine formulated with 101 and
pCD40L should be tested in bovines since this formulation
likely induces protection against BoHV-1 in these animals.
9
Acknowledgments
The authors thank Dr. Osvaldo Zabal for the valuable
assistance with cell cultures, Seppic for providing the
Montanide ESSAI 1113101PR adjuvant, Dr. Kipps for pro-
viding the pDNA3.1-mCD40L in this study, and Ms. Pamela
Angeletti and Fátima Torales for their technical assistance.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This work was supported by INTA Project PNBIO
1131032 and by a grant from Agencia Nacional de Pro-
moción Cientı́fica y Tecnológica PICT2014-0891.
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Address correspondence to:
Dr. Patricia I. Zamorano
Instituto de Virologı́a-IVIT
(INTA-CONICET)
De las Cabañas y De Los Reseros s/n
Hurlingham, Buenos Aires, 1686
Argentina
Buenos Aires
Argentina
E-mail: zamorano.patricia@inta.gob.ar
Dr. Cecilia A. Langellotti
Instituto de Virologı́a-IVIT
(INTA-CONICET)
De las Cabañas y De Los Reseros s/n
Hurlingham, Buenos Aires, 1686
Argentina
Buenos Aires
Argentina
E-mail: langellotti.cecilia@inta.gob.ar